The present invention relates generally to the art of separating charged molecular species, and, in particular, to separation media that are used for capillary electrophoresis.
Gel electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Charged molecular species such as proteins, peptides, nucleic acids and oligonucleotides are separated by causing the species to migrate in a buffer medium under the influence of an applied electric field. The buffer medium normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent, such as for example, agarose or cross-linked polyacrylamide, to promote the separation and to minimize the occurrence of mixing of the species being separated.
Until recently, electrophoretic separations were conducted in gel slabs or open gel beds that were typically fabricated of agarose or cross-linked polyacrylamide material. More recently, capillary electrophoresis (xe2x80x9cCExe2x80x9d) using a polymer gel or solution as a separation medium has been used for the separation of DNA. Capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of charged molecules. Furthermore, capillary electrophoresis can provide quantitative information about a sample using very small amounts of the sample, gel (or polymer solution) and buffer relative to traditional slab gel processes. Moreover, high-resolution separation of charged macromolecules having different effective charges have been achieved.
Typically, the capillary columns used in capillary gel electrophoresis are fabricated from fused silica tubing having diameters on the order of 25 xcexcm to 200 xcexcm and lengths from about 30 cm to about 200 cm. The column interior is filled with buffer and gel separation medium and electrophoretic techniques are used to separate charged molecular species.
Although the pore size of cross-linked polymer gels used for capillary electrophoresis can be controlled by the amount of monomers and cross-linked reagents, polymer gels have been found to be inconvenient as separation media for large scale DNA sequencing analysis due to the instability, irreproducibility and difficulty in controlling the polymerization process inside the capillary tubing.
The inability of many separation media to bind directly to the inner wall of the capillary tubes is a major problem for capillary electrophoresis methods because it creates an electro-osmotic flow when an electric field is applied during electrophoresis. Such migration results in an unsatisfactory separation of the constituent parts of the sample. Traditional methods aimed at preventing electro-osmosis include introducing a compound that binds to the inner surface of a capillary tube wall and that is compatible with the separation medium prior to injecting the separation medium into the tube. For example, U.S. Pat. No. 5,447,617 to Shieh describes covalently bonding polybutadiene to the inner surface of a capillary tube, introducing acrylamide monomers therein and co-polymerizing the acrylamide with the polybutadiene. Such precoating techniques, however, are time consuming, inconvenient and costly.
Another problem encountered in conventional capillary gel electrophoresis results from the use of polyacrylamide-based separation media. Such media are injected into the capillary tube in unpolymerized form. Polymerization of the polyacrylamide is then induced within the capillary tube by well known methods, such as ultraviolet radiation and chemical catalysts. Such methods are characterized by a lack of uniformity in the pore size distribution of the polymer network formed, and by incomplete polymerization.
The irreversible nature of the polymerized polyacrylamide gel also causes numerous problems when a polyacrylamide-based separation medium is used in capillary gel electrophoresis methods. Once the polyacrylamide is polymerized within a capillary electrophoresis tube, the polymerized gel cannot be easily removed from the capillary tube after electrophoresis.
Capillary electrophoresis (xe2x80x9cCExe2x80x9d) provides numerous advantages over conventional slab gel electrophoresis for DNA separation. The use of fused silica capillaries with inner diameters of less than 100 xcexcm enables CE to operate at very high separation voltages and offers fast separation, high efficiency and increased resolution. In addition, rigid gels, which are normally used in slab gel electrophoresis because of their anti-convection ability, are not needed in capillary electrophoresis. Cross-linked polyacrylamide (xe2x80x9cPAMxe2x80x9d) gels, which are widely used in conventional slab gel electrophoresis, were initially used in capillary electrophoresis. Despite successful results, i.e., 700 bases read lengths with resolution of 0.5 for DNA sequencing in about 230 minutes, PAM gels have encountered problems due to bubble formation, gel inhomogeneity, and short lifetime of the capillary.
Accordingly, attempts have been made to use nonpolymerized separation media for capillary electrophoresis. For example, U.S. Pat. No. 5,468,365 to Menchen et al. describes an electrophoresis medium having a matrix of aggregated copolymers dispersed in an aqueous in medium. The polymer matrix of the ""365 patent is described as a dispersion of one substance (micelles) in another substance (water). In such a dispersion, the particles are formed by the association or aggregation of molecules having both hydrophilic and hydrophobic regions. The copolymers of the ""365 patent form a polymer matrix having a relatively uniform mesh size which is believed to be related to the regular, i.e., substantially uniform spacing between adjacent hydrophobic polymer segments.
A number of different polymers have been used in CE methods to separate DNA fragments. Many of these polymers are modified polysaccharides, such as, agarose, methylcellulose (xe2x80x9cMCxe2x80x9d), hydroxypropyl-methyl-cellulose (xe2x80x9cHPMCxe2x80x9d), hydroxyethylcellulose (xe2x80x9cHECxe2x80x9d), hydroxypropylcellulose (xe2x80x9cHPCxe2x80x9d), glucomannan, galactonmannan, and dextran. Some of them are synthesized polymers, such as, polyethylene glycol (xe2x80x9cPEGxe2x80x9d), polyethylene oxide (xe2x80x9cPEOxe2x80x9d), polyvinylpyrrolidone (xe2x80x9cPVPxe2x80x9d), polyvinylalcohol (xe2x80x9cPVAxe2x80x9d), polyacrylamide (xe2x80x9cPAMxe2x80x9d), poly-N-acryloyl-amino-ethoxyethanol (xe2x80x9cPAAEExe2x80x9d), polyacryloylaminopropanol (xe2x80x9cPAAPxe2x80x9d), poly-N,N-dimethylacrylamide (xe2x80x9cPDMAxe2x80x9d), polyacrylamide-co-allyl-xcex2-D-glucopyranoside (xe2x80x9cP(AM/AG)xe2x80x9d), and poly-N-(acryloylaminoethoxyethyl-xcex2-D-glucopyronoside (xe2x80x9cPAEGxe2x80x9d). Recently, polymers with viscosity dependent behavior have also been employed. One type of polymer was characterized by the collapse of molecules at high temperature, such as a copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (xe2x80x9cP(DMA/DEA)xe2x80x9d), a copolymer of poly(N-isopropylacrylamide) (xe2x80x9cPNIPAMxe2x80x9d) densely grafted with short poly(ethylene oxide) (xe2x80x9cPEOxe2x80x9d) chains (xe2x80x9cPNIPAM-g-PEOxe2x80x9d), etc. Other polymers involved the formation of micelles, such as fluorocarbon end-capped polyethylene glycols, E99P69E99 (with E being polyoxyethylene and P being polyoxyproplene), and n-dodecane-poly(ethylene oxide)-n-dodecane, etc. Each of these polymers has distinct advantages, but each also has inherent problems. For example, only several of them, such as HEC, PEO, PVP, PAM, PDMA, P(DMA/DEA), and fluorocarbon end-capped polyethylene glycols, have ever been used for DNA sequencing; and only HEC and high molecular weight PAM, PEO and PDMA have ever achieved a read length of greater than 500 bases.
Entangled polymer solutions, such as liquefied agarose, poly(acrylamide) (xe2x80x9cPAMxe2x80x9d), different kinds of cellulose, poly(ethylene oxide) (xe2x80x9cPEOxe2x80x9d) or poly(dimethylacrylamide), have been widely used as a DNA separation medium in CE with some success. High molecular weight (Mw) PAM has achieved 1,000-base read length in one run for single-stranded DNA in run times of less than one hour. However, the PAM solution has two disadvantages: the injection is very difficult due to the very high viscosity, and the capillary inner wall has to be coated.
One base pair (xe2x80x9cbpxe2x80x9d) resolution of double-stranded DNA has been achieved by using PEO mixtures without coating the capillary inner wall, but the solution viscosity is too high for easy injection. In addition, the capillary channels have to be pretreated with a low viscosity PEO solution before the high viscosity matrix is injected. PEO mixtures have also achieved 1,000 base read length with run times of about seven hours. Other polymers that have been used for single-stranded DNA are poly(dimethylacrylamide) which has achieved about a 600-base read length with run times of about two hours and HEC which has achieved up to about 500 bases with run times of about one hour. A number of other polymers have been used which have achieved read lengths of less than 500 bases.
Mixtures of the same polymer, such as PEO, HEC and PAM, with different molecular weights and mixtures of two modified polysaccharides, i.e., agarose and HEC, have been found to produce a better resolution for both small and large DNA fragments. However, a mixture of two polymers with totally different chemical structures has never been successfully used. Kim et al. (Kim, Y., Yeung, E. S., J. Chromatogr. A., 1997, 781, 315-325) tried to use a mixture of PEO and HPC for DNA sequencing and found the separation to be very poor. The failure was attributed to the incompatibility of the two polymers
The selection of the polymer used as the medium for DNA separation by capillary electrophoresis is very important because the polymer determines the migration behavior and the resolution of DNA fragments. An equally important issue in DNA separation by capillary electrophoresis is the coating of the inner wall of a fused silica capillary, which suppresses both electro-osmosis and the adsorption of DNA fragments onto the capillary inner wall. A coating protocol using PAM covalently attached to the capillary through a Sixe2x80x94Oxe2x80x94Si bond has been widely used. Several modifications of this protocol using PVA, PAAEE, PAAP or PDMA have also been used. Because of the hydrolysis tendency of the Sixe2x80x94Oxe2x80x94Si bond in an alkaline environment, PAM, PAAEE, PAAP or PDMA have even been covalently attached to the capillary by a Sixe2x80x94C bond. Some commercially available GC capillaries from J and W Scientific Inc. with coatings such as DB-1 (100% dimethylpolysiloxane), DB-17 (50% diphenyl 50% dimethylpolysiloxane) and DB-Wax (100% polyethyleneglycols) have also been used. Despite the wide use of covalently coated capillaries, the coating methods often increase their cost by requiring in situ synthesis and give rise to problems such as capillary fouling, coating inhomogeneity, and limited lifetime.
Speed is also important in DNA separation. In the past, pBR322/Hae III has been separated with PAM, PAAEE and PAAP in about one hour. By modifying PAM to have less viscosity, shorter separation times of about tens of minutes have been achieved. With cellulose derivatives and PEO, the separation time is about twenty to thirty minutes. A 13-min separation of pBR322/Hae III has been achieved using the present invention with a separation medium of PNIPAM-g-PEO. Muller et al. has achieved an ultra-fast separation of pBR322/Hae III in 30 seconds with 1% MC. To achieve such a separation, a shorter effective separation length of 3 cm, a higher applied electric field strength of 800 V/cm and a narrower plug electrokinetic injection of 100 ms at 600 V/cm with a fast ramp power supply were employed. However, the separation of 434, 458 and 504 bp was hardly visible. And the partly separated 123/124 bp was a result of the structurally dependent migration at high electric field strengths rather than a result of single base pair resolution.
PAM gels have become less popular due to bubble formation, gel inhomogeneity, and short lifetime of the capillary. These problems have been eliminated in the present invention through the use of non-crosslinked polymer solutions. These polymer solutions can be replaced after each electrophoresis run if necessary, which makes CE particularly well suited for automation.
Accordingly, it would be desirable to provide a new separation medium for capillary electrophoresis methods, which has alleviated the bubble formation problem and reduced the inhomogeneity of the separation medium. It would also be desirable to provide a separation medium that has increased capillary lifetime and does not require coating of the interior surface of a capillary tube. In particular, it would be desirable to provide a separation medium that provides high resolution and is easy to apply and remove from various apparatus.
The present invention is a polymer solution for the efficient separation of charged macromolecules by electrophoresis that includes a plurality of polymers. These polymers are different, do not phase separate when dissolved in solution and are entangled to form an interpenetrating network. In some embodiments, these polymers are neutral and water-soluble. Preferred polymer solutions of the present invention provide at least a 500-base read length in one run for a single-stranded DNA separation.
At least one of the polymers in the polymer solution is PAM, N-substituted PAM, N,N-disubstituted PAM, modified polysaccharides, PEO, PVP, PVA, PEG, or a random, a graft or a block copolymer based on the backbone monomer segments thereof. The nitrogen substitutes are C1 to C3 alkyl, hydroxyl-substituted C1 to C3 alkyl or methoxy-substituted C1 to C3 alkyl. The random, graft or block copolymer can be EPE-type Pluronics, P(DMA/DEA), PNIPAM-g-PEO or P(AM/AG). The polysaccharides can be liquified agrose, MC, HEC, HPMC, HPC, glucomannan, galactonmannan and dextran.
In a preferred embodiment, at least one of the polymers in the polymer solution is a silica-absorbing polymer that suppresses electrophoendoosmotic flow and charged macromolecule-silica interactions. The silica-absorbing polymer can be PVP, PEO, EPE-type Pluronics, N-substituted PAM or N,N-disubstituted PAM. The nitrogen substitutes can be C1 to C3 alkyl, hydroxyl-substituted C1 to C3 alkyl, or methoxy-substituted C1 to C3 alkyl.
The interpenetrating network polymer solution has a more expanded structural formation than the entanglement structure of a corresponding homopolymer solution and it has a larger effective size than that of a corresponding homopolymer solution. This represents an effective entanglement network greater than that of the corresponding homopolymers. The interpenetrating network can be prepared by synthesizing a first polymer in a matrix of a second polymer solution or by dissolving together the two polymers in a solvent.
Another embodiment of the present invention is a polymer solution for the efficient separation of charged molecules by electrophoresis that can provide at least a 500-base read length in one run for a single-stranded DNA separation. The polymer solution includes a plurality of stretched polymer chains that have polymer chain entanglement times greater than the corresponding linear homopolymer solution. The polymer chains include the same polymer or a plurality of different polymers. These polymer chains can entangle to form an interpenetrating network in solution. Preferred polymers are PAM and PVP or PDAM and PVP.
The polymer chains can include a random copolymer made up of a monomer taken from PAM, N-substituted PAM, N,N-disubstituted PAM, modified polysaccharides, PEO, PVP, PVA or PEG. The random copolymer can also include a silica-absorbing segment taken from PVP, PEO, EPE-type Pluronics, N-substituted PAM or N,N-disubstituted PAM, wherein nitrogen substitutes can be C1 to C3 alkyl, hydroxyl-substituted C1 to C3 alkyl, or methoxy-substituted C1 to C3 alkyl. In a preferred embodiment, the random copolymer is made up of AM and DMA.
The polymer chains can include a graft copolymer that includes a monomer taken from PAM, N-substituted PAM, N,N-disubstituted PAM, modified polysaccharides, PEO, PVP, PVA or PEG. The graft copolymer can also include a silica-absorbing segment taken from PVP, PEO, EPE-type Pluronics, N-substituted PAM or N,N-disubstituted PAM, wherein nitrogen substitutes can be C1 to C3 alkyl, hydroxyl-substituted C1 to C3 alkyl, or methoxy-substituted C1 to C3 alkyl. In a preferred embodiment, the graft copolymer includes PNIPAM-g-PEO.
The polymer chains can include a very weakly cross-linked microgel that includes a monomer taken from PAM, N-substituted PAM, N,N-disubstituted PAM, modified polysaccharides, PEO, PVP, PVA or PEG. The very weakly cross-linked microgel can also include a silica-absorbing segment taken from PVP, PEO, EPE-type Pluronics, N-substituted PAM or N,N-disubstituted PAM, wherein nitrogen substitutes can be C1 to C3 alkyl, hydroxyl-substituted C1 to C3 alkyl, or methoxy-substituted C1 to C3 alkyl. In a preferred embodiment, the very weakly cross-linked microgel includes PAM and a hydrophilic cross-linker, preferably PEO diacrylate.
The polymer solution of the present invention satisfies the need for improved DNA separation media that can also be used to dynamically coat the inner capillary wall. In addition, the separation media of the present invention are easier to use because they are not strongly cross-linked and they provide improved resolution and faster run times.